Mechanisms of Normal Tissue Radiation Responses Lecture 18 Mechanisms of Normal Tissue Radiation Responses

Differences between slowly and rapidly proliferating tissues Molecular and cellular responses in slowly and rapidly proliferating tissues Cytokines and growth factors Regeneration Remembered dose Functional subunits: Mechanisms underlying clinical symptoms Latency Inflammatory changes Cell killing: Radiation fibrosis and vascular damage Volume effects: Pharmacological modification of normal tissue responses

Differences between slowly and rapidly proliferating tissues Cell death after irradiation occurs mostly as cells attempt to divide. In tissues with a rapid turnover rate, damage becomes evident quickly: in a matter of hours in the intestinal epithelium and bone marrow, in a matter of days in the skin and mucosa. They are rapidly dividing self-renewal tissues; The spinal cord, and kidney, by contrast, are late-responding tissues. The current philosophy: radiation damage to the tissue is a function simply of different cell-turnover rate.

Differences between slowly and rapidly proliferating tissues Law of Bergonie and Tribondeau (1906): Tissues appear to be more “radiosensitive” if their cells are less-well differentiated, have a greater proliferative capacity, and divide more rapidly.

Differences between slowly and rapidly proliferating tissues Note the substantial range of radio-sensitivities with shoulder width being the principal variable

Differences between slowly and rapidly proliferating tissues The radio-sensitivity of cells from normal tissues varies widely. The width of the shoulder of the curve is the principal variable.

Differences between slowly and rapidly proliferating tissues Crypt cells of the mouse jejunum have a very large shoulder

Differences between slowly and rapidly proliferating tissues Bone-marrow stem cells have little if any shoulder. Most other cell types fall in between

Differences between slowly and rapidly proliferating tissues All normal tissues are not the same. There is a clear distinction between tissues that are early responding, such as the skin, mucosa, and intestinal epithelium, and those that are late- responding, such as spinal cord. Figure shows the extra dose required to produce a given level of damage from the early- and late-responding tissues.

Differences between slowly and rapidly proliferating tissues An attempt to convert the experimental laboratory data (previous slide) into a general principal that can be applied to clinical practice

Differences between slowly and rapidly proliferating tissues The shape of the dose-response relationship for early- and late-responding tissues.

Differences between slowly and rapidly proliferating tissues In experiments with laboratory animals the isoeffect curves (e.i., dose versus number of fractions to produce an equal biologic effect) are steeper for a range of late effects than for a variety of acute effects

Differences between slowly and rapidly proliferating tissues A summary of the values of α/β for a number of early- and late-responding tissues

Differences between slowly and rapidly proliferating tissues Possible explanations for the difference in shape of dose-response relationships for early- and late-responding tissues: Radiosensitivity varies with the cell cycle. In general, cells are most resistant in late S phase; slowly growing cells with a long cycle, however, may have a second resistant phase in the early G1 phase, which may be termed Go if the cells are out of cycle. Thus, two different cell populations may be radioresistance: a population proliferating so fast that S phase occupies a major portion of the cycle; a population proliferating so slowly that many cells are in early G1 or not proliferating at all, so many resting cells are in Go

Differences between slowly and rapidly proliferating tissues Possible explanations for the difference in shape of dose-response relationships for early- and late-responding tissues: It is thought that many late-responding normal tissues are resistant, owing to the presence of many resting cells. If resistance results from the presence of many cells in S phase in a rapidly proliferating population, redistribution occurs through all the phases of the cell cycle-”self-sensitizing” activity. This applies to acutely responding tissues and also to tumors.

Differences between slowly and rapidly proliferating tissues Molecular and cellular responses in slowly and rapidly proliferating tissues Cytokines and growth factors Regeneration Remembered dose Functional subunits: Mechanisms underlying clinical symptoms Latency Inflammatory changes Cell killing: Radiation fibrosis and vascular damage Volume effects: Pharmacological modification of normal tissue responses

Molecular and cellular responses in slowly and rapidly proliferating tissues Let us first consider the normal tissues which are not subject to mutations and other acquired changes that might affect radiosensitivity. What are there molecular differences that underlie the different α/β ratios between slowly proliferating tissue such as the kidney and acutely proliferating tissue such as mucosis? The most striking difference is the proliferation rate. The proliferation rate will influence the cell-cycle distribution of clonogenic cells, and in this way may indirectly affect the tissue α/β ratio. The lethal effects of ionizing radiation are thought to be due to DNA double strand breaks (DSBs) in both acutely and late responding tissues.

Molecular and cellular responses in slowly and rapidly proliferating tissues The two main pathways that repair DSBs are the end joining (EJ) repair pathway and the homologous recombination (HR) repair pathway. In human cells, homologous recombination can only occur after replication has occurred and thus when a sister chromatid is available, namely in G2 and S phase. Therefore it seems reasonable to hypothesize that, homologous recombination may be more prominent in the fast proliferating tissues. If this is the case, then the tissues with a high α/β ratio are also the same which have higher amounts of homologous recombination and which are spared less by fractionation. Since the homologous recombination pathway is characterized by a very long repair halftime, in some instances of more than 24 hours, the repair of DNA damage between fractions may be less complete in these tissues. In other words, the sparing effect of fractionation may be smaller in fast proliferating tissues as a consequence of the long repair halftimes for homologous recombination.

Molecular and cellular responses in slowly and rapidly proliferating tissues What is the situation in tumors? If the same reasoning is used as in normal tissue, we would expect that slowly proliferating tumors would have a low α/β ratio due to the reduced importance of homologous recombination. Similarly, rapidly proliferating tumors would have a comparatively higher α/β ratio. This interpretation is complicated by the fact that tumors have a wide variation in the proliferation rate both among and within tumors. A further complication is the increasing evidence suggesting that many tumors acquire defects in one of several DNA repair pathways. DNA repair acts as a tumor suppressor and its inactivation can thus accelerate tumor formation. This is especially relevant for documented clinical defects in genes involved in homologous recombination such as:     MRE11, RAD51,52,54, XRCC2,3, RAD51B,C,D, MRE11 / NBSI / RAD50, BRCA1,2, ATM.

Molecular and cellular responses in slowly and rapidly proliferating tissues For example we would predict that tumors with a defect in homologous recombination, such as BRCA1 tumors (see figure), should have a comparatively low α/β ratio even if they are rapidly proliferating. (Many gene defects have been studied in the DT40 cell line (chicken).) At this point the suggestion that increased homologous recombination in tumors and normal tissues results in a higher α/β ratio remains a hypothesis which needs to be tested experimentally. However, if it is true it would suggest that analysis of proliferation and DNA repair defects could be used to rationally choose fractionation schedules. It would certainly be interesting to determine if the recent data suggesting that prostate cancer has a low α/β ratio could be explained by either their known slow proliferation rate and/or a defect in homologous recombination and/or a well oxygenated status. BRCA1 is required for transcription-coupled repair of oxidative DNA damage. Effect of BRCA1 deficiency on survival after radiation. ■ wild-type, ▼ Brca1 positive, ● Brca1 deficient, ¨ Brca1 deficient cell line. From Gowen et al. Science, 281: 1009, 1998.

Molecular and cellular responses in slowly and rapidly proliferating tissues Ionizing radiation can act to regulate the expression of a number of genes including early response genes such as c-fos, c-jun, and c-myc, cell-cycle control genes, genes involved in DNA repair and apoptosis, cytokines and growth factors.

Differences between slowly and rapidly proliferating tissues Molecular and cellular responses in slowly and rapidly proliferating tissues Cytokines and growth factors Regeneration Remembered dose Functional subunits: Mechanisms underlying clinical symptoms Latency Inflammatory changes Cell killing: Radiation fibrosis and vascular damage Volume effects: Pharmacological modification of normal tissue responses

Cytokines and growth factors Radiation induces interleukins 1 (IL-1) as well as 6 (IL-6). IL-1 acts as a radioprotectant of hematopoietic cells by increasing both the shoulder and D0 of the survival curve. Basic fibroblast growth factor induces endothelial cell growth, inhibits radiation-induced apoptosis thus protecting against microvascular damage. Platelet derived growth factor β increases damage to vascular tissue. Transforming growth factor beta induces a strong inflammatory response. It may down-regulate IL-1 and TNF and increase damage to hematopoietic tissue. Tumor necrosis factor (TNF) mediates the inflammatory response produced by monocytes and tumor cells by binding to cell-surface receptors that initiate signal-transduction pathways.

Extracellular Control of Growth and Division Within the context of a multicellular organism, extracellular signals are critical to coordinate cell growth, division and death. The size of an organ or organism depends on the balance and coordination of the three processes of cell growth, division and death. Cell growth, division and death often are independently regulated. Extracellular signals that regulate these processes are proteins that may be soluble, bound to the cell surface or bound to the extracellular matrix.

There are Three Classes of Signals That Regulate Division, Growth and Death Mitogens stimulate cell division primarily through relieving intracellular negative controls. Growth factors stimulate cell growth primarily by promoting synthesis of macromolecules. Survival factors promote cell survival by suppressing apoptosis. Single agents often have overlapping mitogen, growth factor and survival factor activities.

Mitogens Stimulate G1 and G1/S-Cdks

Mitogens Stimulate G1 and G1/S-Cdks Examples of mitogens are PDGF and EGF. Some factors like TGF act as mitogens in some cells and inhibitors of division in others. Many mitogens have additional activities such as growth factors, survival factors, or differentiation factors, depending on the cell type and circumstances.

Growth Factors Often Work Through PI-3 Kinases to Increase the Number of Ribosomes Nerve growth factor and other related neurotrophins are example of growth factors. Growth factors often have multiple activities, including mitogen and cell survival factor activity.

Ionizing radiation Primary Messengers Second Messengers Membrane phospholipid changes Ligand-receptor binding Primary Messengers Cell Membrane Activated relay molecule (GTPase) Kinase activation Second Messengers Cytosol Activated proteins Inactivation of proteins (ubiquitination) Precursor gene events Translocation of transcription factors Induction / repression of gene expression Changes in gene expression Nucleus

Radiation-Induced Apoptosis Early sensors Mediators Effectors P53 Ceramide Bax Bid cleavage Dephosphorylation of Bad Par-4 PUMA TRADD/FADD ATM activation TNF-a EGR-1 p53 Cleavage of pro-caspases Endonucleases Smac/DIABLO Release of Ctychrome C Apaf-1 PARP cleavage Cleavage of cytoskeleton proteins Gene signaling events Ionizing radiation Chromatin condensation DNA Fragmentation Morphological changes Chromatin condensation Phosphatidylserine externalization Membrane Blebbing Cell shrinkage Apoptotic bodies

Signaling that activate p53 DNA damage (double strand breaks) Aberrant growth signals (such as overactivation of ras or myc) Drugs (chemotherapeutics) and UV light All 3 pathways inhibit the degradation of p53, allowing it to activate transcription of genes that induce cell death or inhibit cell division.

p53 mediated transcriptional activation of apoptosis “Death receptors” Some p53 target genes: BCL-2, BCL-XL : block the release of cytochrome c BAX, BAK: inhibit BCL-2 and BCLXL

Differences between slowly and rapidly proliferating tissues Molecular and cellular responses in slowly and rapidly proliferating tissues Cytokines and growth factors Regeneration Remembered dose Functional subunits: Mechanisms underlying clinical symptoms Latency Inflammatory changes Cell killing: Radiation fibrosis and vascular damage Volume effects: Pharmacological modification of normal tissue responses

Regeneration Replacement of damaged cells in the organ by the same cell type present before radiation; also called primary repair. Healing of a tissue or organ occurs by one of two means: regeneration, the replacement of damaged cells in the organ by the same cell type present before radiation; or repair, the replacement of the depleted original cells by a different cell type. Regeneration results in a total or partial reversal of early radiation changes, actually restoring the organ to its pre-irradiated state both morphologically and functionally. In this situation, any chronic changes would be of a primary nature, I.e., due to depletion of a different cell type in a different part of the tissue.

Regeneration In normal tissues the time of onset of regeneration varies between tissues, depending on the cellular proliferation rate, because radiation-induced cell death is linked to mitosis. Tissues with high turnover rate (acutely responding), such as intestine and mucosa, will show compensatory proliferation during a fractionated course of radiotherapy, while those with more slowly proliferating cells (late responding), such as kidney and spinal cord, will exhibit little, if any, regenerative response during fractionated radiotherapy. Protracting the overall treatment time will allow regeneration of surviving clonogenic cells and will be maximal in rapidly dividing tissue, but will be considerably less (if occurs at all) in slowly proliferating tissues.

Regeneration Time as a factor Acutely responding normal tissues will show changes sooner than the late responding organs exposed to the same dose. However, at a later times the late responding tissues may manifest more severe response. For example, irradiation of one lung (late responding) and the overlying skin (acutely responding) with a single dose of 20 Gy produces marked changes in the skin at 6 months postexposure,but lung changes are less severe. Observations at 1 year postexposure reveal minimal skin changes, but now the irradiated lung exhibits severe changes.

Differences between slowly and rapidly proliferating tissues Molecular and cellular responses in slowly and rapidly proliferating tissues Cytokines and growth factors Regeneration Remembered dose Functional subunits: Mechanisms underlying clinical symptoms Latency Inflammatory changes Cell killing: Radiation fibrosis and vascular damage Volume effects: Pharmacological modification of normal tissue responses

“Remembered Dose” Wondergem J, Haveman J. (1983) The response of previously irradiated mouse skin to heat alone or combined with irradiation: influence of thermotolerance. Int J Radiat Biol Relat Stud Phys Chem Med, 44, 539-52.Wondergem J, Haveman J. (1983) The response of previously irradiated mouse skin to heat alone or combined with irradiation: influence of thermotolerance. Int J Radiat Biol Relat Stud Phys Chem Med, 44, 539-52. The skin of the mouse foot was used to study the effects of previous irradiation on the response to hyperthermia (44 degrees C), to irradiation, or to irradiation combined with hyperthermia (43 degrees C or 44 degrees C). Hyperthermia was applied by immersing the mouse foot into a hot waterbath and irradiation was performed using a 250 kV X-ray generator. Previous irradiation of the feet of mice 90 days before, with 20 Gy, increased the subsequent response to heat alone, or when combined with irradiation, as well as to irradiation alone. It had little effect on the thermal enhancement ratio's for both acute and late skin reactions. Memory of the previous irradiation treatment could be masked when the temperature of the subsequent heat treatment alone, or when combined with irradiation, was 44 degrees C. A priming heat treatment induced resistance both to a subsequent heat treatment and to a subsequent combined irradiation-heat treatment in normal skin as well in previously irradiated skin. This 'resistance' is probably mainly the result of thermotolerance induced in cells in the skin by the priming heat treatment. In thermotolerant skin a 'memory' of the previous irradiation was always evident when the reaction after heat alone or heat combined with irradiation was measured. When the late skin reaction was considered, a larger 'memory' of the previous irradiation treatment was always evident, compared to the acute skin reaction: the 'remembered' dose in the late skin reaction was about two times the 'remembered dose' in the acute skin reaction.

Differences between slowly and rapidly proliferating tissues Molecular and cellular responses in slowly and rapidly proliferating tissues Cytokines and growth factors Regeneration Remembered dose Functional subunits: Mechanisms underlying clinical symptoms Latency Inflammatory changes Cell killing: Radiation fibrosis and vascular damage Volume effects: Pharmacological modification of normal tissue responses

Functional subunits: Mechanisms underlying clinical symptoms The relationship between the survival of clonogenic cells and organ function, or failure, depends on the structural organization of the tissue. Many tissues may be thought of as consisting of functional subunits (FSUs). In some tissues, the FSUs are discrete-nephron in the kidney, the lobule in the liver, acinus in the lung; In other tissues FSUs have no clear anatomical demarcation-the skin, the mucosa, and the spinal cord. The response to radiation of these two types of tissue-with structurally defined or structurally undefined FSUs-is quite different.

Functional subunits: Mechanisms underlying clinical symptoms Survival of structurally defined FSUs depends on the survival of one or more clonogenic cells within them. Tissue survival then will depend on the number and radiosensitivity of these clonogens. Surviving clonogens cannot migrate from one to the other. Because each FSU is both small and autonomous, low doses deplete the clonogens in it. Example:low tolerance of kidney to radiation. By contrast, the clonogenic cells that can repopulate the structurally undefined FSUs after depletion by radiation are not confined to one particular FSU itself. Clonogenic cells can migrate and allow repopulation of a depleted FSU. Example: re-epithelization of a denuded area of skin by migration from adjacent areas.

Differences between slowly and rapidly proliferating tissues Molecular and cellular responses in slowly and rapidly proliferating tissues Cytokines and growth factors Regeneration Remembered dose Functional subunits: Mechanisms underlying clinical symptoms Latency Inflammatory changes Cell killing: Radiation fibrosis and vascular damage Volume effects: Pharmacological modification of normal tissue responses

Latency Example: Spinal cord myelopathy Over a dose range of about 25 to 60 Gy, delivered in single doses, The general tendency is a decreasing latency with increases in dose of approximately 2days/Gy. There is a considerable variation with animal strain, as well as with the region of the cord irradiated. Mechanisms: demyelination or slowly progressive atrophy is probably a consequence of interference with the slow continuous turnover of oligodendrocytes by killing of glial progenitor cells. Vascular injury may accelerate, precipitate, or even initiate the white-matter changes leading to necrosis.

Latency Example: Spinal cord myelopathy A dose-response relationship can be determined for late damage caused by local irradiation of the spinal cords of rats. After latent periods of 4 to 12 months, symptoms of myelopathy develop, followed some time later by impaired use of the hind legs. The following slide shows the steep dose-response relationship for hind-limb paralysis following the irradiation of the section of the spinal cord in rats.

Latency Spinal cord myelopathy

Latency Spinal cord myelopathy Various syndromes of radiation-induced injury in rodent brain and spinal cord are very similar to those described in humans: Lesions observed in first 6 months are limited to the white matter; The most common type of late delayed injury peks at 1 to 2 years postirradiation and has a vascular basis; another type of late injury is slowly progressive glial atrophy. Over a dose range of about 25 to 60 Gy delivered in single doses, the general tendency is a decreasing latency with increases in dose of approximately 2 days/Gy.

Differences between slowly and rapidly proliferating tissues Molecular and cellular responses in slowly and rapidly proliferating tissues Cytokines and growth factors Regeneration Remembered dose Functional subunits: Mechanisms underlying clinical symptoms Latency Inflammatory changes Cell killing: Radiation fibrosis and vascular damage Volume effects: Pharmacological modification of normal tissue responses

Inflammatory changes Expression of pro-inflammatory cytokines IL-1 anf TNF-alpha by irradiated endothelial cells; Expression of COX I and COX II is linked to local inflammatory responses; Enhanced interaction of leukocytes with endothelial monolayers, and with blood vessel wall of irradiated normal tissues; Two other molecules that are potentially implicated in the enhanced adhesion of leukocytes are platelet activating factor (PAF) and P-selectin. These molecules appear to act in concert to mediate the earliest adhesion events. Their expression is stimulated by ROI and radiation enhances their production; Radiation up-regulates the expression of the E-selectin gene, which is known to contain NF-kappa B and AP-1 sites in its promoter region.

Differences between slowly and rapidly proliferating tissues Molecular and cellular responses in slowly and rapidly proliferating tissues Cytokines and growth factors Regeneration Remembered dose Functional subunits: Mechanisms underlying clinical symptoms Latency Inflammatory changes Cell killing: Radiation fibrosis and vascular damage Volume effects: Pharmacological modification of normal tissue responses

Radiation fibrosis and vascular damage

Radiation fibrosis and vascular damage It is logical to assume that if one or more of the pathways leading from radiation to fibrosis could be blocked, the injury caused by radiation could be reduced or eliminated. Several pharmaceuticals have been used to attempt to block the changes

Radiation fibrosis and vascular damage Examples of the results of these blocking agents on postradiation changes in vascular permeability and blood flow.

Differences between slowly and rapidly proliferating tissues Molecular and cellular responses in slowly and rapidly proliferating tissues Cytokines and growth factors Regeneration Remembered dose Functional subunits: Mechanisms underlying clinical symptoms Latency Inflammatory changes Cell killing: Radiation fibrosis and vascular damage Volume effects: Pharmacological modification of normal tissue responses

Volume effects It is generally observed in clinical radiotherapy that the total dose that can be tolerated depends on the volume of tissue irradiated. Tolerance dose has been defined as that dose that produces an acceptable probability of a treatment complication. The spatial arrangement of the SFUs in the tissue is critical.

Volume effects Figure illustrates the effect on the probability of development of a threshold- binary or quantum response in a serial FSU tissue. By contrast, tissues in which FSUs are not arranged serially show a graded dose response and do not show a volume effect at lower levels of injury. This is true for skin or mucosa.

Volume effects The total volume of irradiated tissue is assumed to have an influence on the development of tissue injury. The spinal cord is the clearest case in which the functional subunits (FSUs) are arranged in linear fashion like links in a chain.